Secreted and cell surface genes expressed in benign and malignant colorectal tumors

ABSTRACT

Serial analysis of gene expression (SAGE) was used to identify transcripts encoding secreted or cell-surface proteins that were expressed in benign and malignant tumors of the colorectum. A total of 290,394 tags were analyzed from normal, adenomatous and cancerous colonic epithelium. Of the 21,343 different transcripts observed, 957 were found to be differentially expressed between normal and adenoma or between normal and cancer. Forty-nine transcripts were elevated ≧20-fold in adenomas, 40 transcripts were elevated ≧20-fold in cancers, and nine transcripts were elevated ≧20-fold in both. Product of six these nine transcripts (TGFBI, LYS, RDP, MIC-1, REGA, and DEHL) were predicted to be secreted or to reside on the cell surface and these were analyzed in more detail. The abnormal expression levels predicted by SAGE were confirmed by quantitative PCR analyses of each of these six genes. Moreover, the cell types responsible for the elevated expression were identified by in situ hybridization and by PCR analyses of epithelial cells immunoaffinity purified from primary tumors.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field of the Invention

[0002] The invention relates to the early detection of colorectaladenoma and carcinoma. In particular it relates to the detection ofsecreted or cell surface markers in easily collectible bodily samples.

[0003] 2. Background of the Art

[0004] Colorectal cancer is the second leading cause of cancer death inthe United States, with ˜130,000 patients diagnosed each year and‘50,000 ultimately succumbing to the disease (1). Most colorectalcancers develop slowly, beginning as small benign colorectal adenomaswhich progress over several decades to larger and more dysplasticlesions which eventually become malignant. This gradual progressionprovides multiple opportunities for prevention and intervention. Indeed,benign adenomas can be detected and removed by simple colonoscopy andpolypectomy, precluding the need for radical surgical and adjuvanttreatments. It is therefore believed that early detection and removal ofthese benign neoplasms provides the best hope for minimizing morbidityand mortality from colorectal cancer. Various screening methods fordetecting early colorectal tumors are available, such as fecal occultblood testing, sigmoidoscopy, and colonoscopy (reviewed in 2). However,none of these methods are optimal, and new approaches are needed.

BRIEF SUMMARY OF THE INVENTION

[0005] In a first embodiment a method is provided for detection ofcolorectal adenoma and carcinoma. An mRNA sample is isolated from fecesof a subject. Renal dipeptidase mRNA in said mRNA sample is detected.The amount of renal dipeptidase mRNA in said mRNA sample is compared toamounts of renal dipeptidase mRNA in normal subjects. An elevated amountof renal dipeptidase mRNA in said mRNA sample is an indicator ofcolorectal adenoma or carcinoma in the subject.

[0006] According to another embodiment of the invention a method isprovided for detection of colorectal adenoma or carcinoma. Epithelialcells are isolated from blood of a subject. An mRNA sample is isolatedfrom the epithelial cells. Renal dipeptidase mRNA in said mRNA sample isdetected. The amount of renal dipeptidase mRNA in said mRNA sample iscompared to amounts of renal dipeptidase mRNA in normal subjects. Anelevated amount of renal dipeptidase mRNA in said mRNA sample is anindicator of colorectal adenoma or carcinoma in the subject.

[0007] A third embodiment of the invention provides a method fordetection of colorectal adenoma or carcinoma. Blood of a subject iscontacted with a renal dipeptidase substrate. Activity of renaldipeptidase in said blood is determined by detection of increasedreaction product or decreased renal dipeptidase substrate. The amount ofactivity of renal dipeptidase in blood of the subject is compared tothat in normal subjects. An elevated amount of activity of renaldipeptidase in the blood of the subject is an indicator of colorectaladenoma or carcinoma in the subject.

[0008] According to another embodiment of the invention a method fordetection of colorectal adenoma or carcinoma is provided. Feces of asubject is contacted with a renal dipeptidase substrate. Activity ofrenal dipeptidase in said feces is determined by detection of increasedreaction product or decreased renal dipeptidase substrate.

[0009] The amount of activity of renal dipeptidase in feces of thesubject is compared to that in normal subjects, wherein an elevatedamount of activity of renal dipeptidase in the feces of the subject isan indicator of colorectal adenoma or carcinoma in the subject.

[0010] Another embodiment of the invention provides a method fordetection of colorectal adenoma or carcinoma. An antibody isadministered to a subject. The antibody specifically binds to renaldipeptidase and is labeled with a moiety which is detectable fromoutside of the subject. The moiety in the subject is detected fromoutside of the subject. An area of localization of the moiety within thesubject but outside the proximal tubules of the kidney identifiescolorectal adenoma or carcinoma.

[0011] Another method is also provided for detection of colorectaladenoma or carcinoma.

[0012] An inhibitor of renal dipeptidase is administered to a subject.The inhibitor is labeled with a moiety which is detectable from outsideof the subject. The moiety in the subject is detected from outside ofthe subject. An area of localization of the moiety within the subjectbut outside the proximal tubules of the kidney identifies colorectaladenoma or carcinoma.

[0013] According to yet another method for detection of colorectaladenoma or carcinoma, a substrate for renal dipeptidase is administeredto a subject. The substrate is labeled with a detectable moiety. Fecesare isolated from the subject. Renal dipeptidase reaction product orrenal dipeptidase substrate with the detecable moiety is detected in thefeces. An increased reaction product or decreased reaction substrate inthe feces indicates colorectal adenoma or carcinoma in the subject.

[0014] Still another method for detection of colorectal adenoma orcarcinoma is provided by the present invention. A substrate for renaldipeptidase is administered to a subject.

[0015] The substrate is labeled with a detectable moiety. Blood from thesubject is subsequently isolated. Renal dipeptidase reaction product orrenal dipeptidase substrate with the detecable moiety is detected in theblood. An increased product or decreased substrate in the bloodindicates colorectal adenoma or carcinoma in the subject.

[0016] Still another embodiment of the invention is a method fordetection of colorectal adenoma or carcinoma. Renal dipeptidase in bloodof a subject is detected and compared to the amount of renal dipeptidasein normal subjects. An elevated amount of renal dipeptidase in the bloodof the subject is an indicator of colorectal adenoma or carcinoma in thesubject.

[0017] Still another embodiment of the invention is a method fordetection of colorectal adenoma or carcinoma. Renal dipeptidase in fecesof a subject is detected and compared to the amount of renal dipeptidasein normal subjects. An elevated amount of renal dipeptidase in the fecesof the subject is an indicator of colorectal adenoma or carcinoma in thesubject.

[0018] Yet another embodiment of the invention is a method for detectionof colorectal adenoma or carcinoma. An mRNA sample is isolated fromfeces of a subject.

[0019] Macrophage inhibitory cytokine mRNA is detected in the mRNAsample. The amount of macrophage inhibitory cytokine mRNA in said mRNAsample is compared to amounts of macrophage inhibitory cytokine mRNA innormal subjects. An elevated amount of macrophage inhibitory cytokinemRNA in said mRNA sample is an indicator of colorectal adenoma orcarcinoma in the subject.

[0020] Another embodiment of the invention is a method for detection ofcolorectal adenoma or carcinoma. Epithelial cells are isolated fromblood of a subject. An mRNA sample is isolated from the epithelialcells. Macrophage inhibitory cytokine mRNA is detected in said mRNAsample. The amount of macrophage inhibitory cytokine mRNA in said mRNAsample is compared to amounts of macrophage inhibitory cytokine mRNA innormal subjects. An elevated amount of macrophage inhibitory cytokinemRNA in said mRNA sample is an indicator of colorectal adenoma orcarcinoma in the subject.

[0021] Still another embodiment of the invention is a method fordetection of colorectal adenoma or carcinoma. Macrophage inhibitorycytokine in blood of a subject is detected and compared to the amount ofmacrophage inhibitory cytokine in normal subjects. An elevated amount ofmacrophage inhibitory cytokine in the blood of the subject is anindicator of colorectal adenoma or carcinoma in the subject.

[0022] Still another embodiment of the invention is a method fordetection of colorectal adenoma or carcinoma. Macrophage inhibitorycytokine in feces of a subject is detected and compared to the amount ofmacrophage inhibitory cytokine in normal subjects. An elevated amount ofmacrophage inhibitory cytokine in the feces of the subject is anindicator of colorectal adenoma or carcinoma in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1.A, Distribution of the fold changes of differentiallyexpressed transcript tags. Transcripts in which the significancecriterion was met (p<0.05, a total of 957 tags) in the comparisonsbetween normal and adenoma or normal and cancer are plotted in thefigure. The ratios of adenoma to normal and cancer to normal wereplotted on a log scale. The shaded box in (FIG. 1.A) and enlarged in(FIG. 1.B) encloses the transcript tags detailed in Table 3. The twounlabeled dots correspond to tags whose differential expression couldnot be confirmed by quantitative PCR suggesting that the tags werederived from different transcripts than the ones indicated in Table 3.

[0024]FIG. 2. Quantitative PCR analysis of genes elevated in bothadenomas and cancers. Quantitation of expression of genes in tumors andmatched normal tissues from five patients (Pt) are shown as foldelevation over that in matched normal colonic mucosa. Each barrepresents the average of three independent measurements. TGFBI, LYS,RDP, MIC-1, REGA, and DEHL are as described in Table 3.

[0025]FIG. 3. Quantitative PCR analysis of genes decreased in bothadenomas and cancers. Quantitation of expression of genes in tumors andmatched normal tissue from five patients (Pt) are shown as a fraction ofmatched normal. Each bar represents the average of three independentmeasurements. CA2 and DRA are described in Table 4. Dual SpecificityPhosphatase (DUSPI), and Acid Sphingomylenase-like phosphodiesterase(ASML3a) represented transcripts that were repressed but did not meetthe stringent criteria required for inclusion in Table 4. SAGE dataindicated that DUSP1 was 5- and 76-fold repressed in adenomas andcancers, respectively. ASML3a was 15-fold repressed in both adenoma andcancer.

[0026]FIG. 4. Quantitative PCR analysis of mRNA from purified epithelialcells of genes elevated in both adenomas and cancers. Quantitation ofexpression of genes in the purified normal (N) or cancer (Ca) epithelialcells taken from two patients are shown as fold elevation over matchednormal. Genes examined were the same as in FIG. 2.

[0027]FIG. 5A-FIG. 5E. In-situ hybridization analyses of elevated genes.Genes examined were REGA (FIG. 5A), TGFBI (FIG. 5B), LYS (FIG. 5C), RDP(FIG. 5D), and MIC-1 (FIG. 5E). Positive cells appear red, arrows pointto clusters of malignant epithelial cells, and arrow heads point tomacrophages.

[0028]FIG. 6. Inhibitors of renal dipeptidase demonstrate inhibitionconstants ranging from 0.6 nM to 19.5 nM.

[0029]FIG. 7. A comparison of the inhibitors shown in FIG. 6. comparesthe inhibition rate as a function of concentration of inhibitor.

[0030]FIG. 8. Substrates of renal dipeptidase are shown.

[0031]FIG. 9 shows the difference in activity of renal dipeptidase foundin adenomas, cancer, and metastases compared to normal colonic tissue.

DETAILED DESCRIPTION OF THE INVENTION

[0032] It is a finding of the present invention that particular genesare aberrantly and consistently expressed in both adenomas andcarcinomas of the colon. Products of such genes provide cellular andserum markers for colorectal neoplasia. The ideal tumor marker would beexpected to have several characteristics. First, it should be expressedat high levels in tumors and at greatly reduced levels in normaltissues. Second the elevated expression should occur early and remainelevated during the neoplastic process. Third, such a marker should beelevated in the majority of clinical samples. Fourth, the marker shouldbe cell surface or secreted to facilitate its detection. We haveidentified several genes that appear to meet all of these criteria andmay therefore be especially useful as diagnostic tools for the earlydetection of colorectal neoplasia, even of presymptomatic colorectalneoplasia. Any of the markers identified in Tables 3 and 5 can be used,particularly Renal Dipeptidase and Macrophage Inhibitory Cytokine.

[0033] Serum markers can be found and detected in whole blood, serum,plasma, or fractions thereof. These are collectively referred to as“blood” herein. Markers can also be found in stool. Samples for testingcan be feces or processed or fractionated feces. All such samples arereferred to herein as “feces.”

[0034] Inhibitors of markers which are enzymes, such as RenalDipeptidase, can be used as affinity reagents for labeling the marker.Preferably the inhibitors are those which bind irreversibly.Alternatively they are ones which bind and release, but release at aslow rate. Inhibitors with suitably slow release rates are those whichhave a binding half-life of greater than 30 minutes, or 1, 2, 3, 5, 8,or 10 hours. Many inhibitors of Renal Dipeptidase are known, includingthe commercially available Cilastatin, and phosphinic acid inhibitors.See Parsons et al., “A new class of potent, slowly reversiblydehydropeptidase inhibitors,” Biochemistry International, vol. 23, pp.1107-1115, 1991. Inhibitors which covalently bind to and/or modify RenalDipeptidase are also known and can be used. See Wu and Mobashery,“Targeting renal dipeptidase (dehydropeptidase I) for inactivation bymechanism-based inactivators,” J. Med. Chem., vol. 34, pp. 1914-1916,1991. Some inhibitors mimic transition states between substrates andproduct. Some useful inhibitors are shown in FIG. 6. These includeinhibitors having halogen substitutions. Such inhibitors can be readilymade using radioactive halogens for ready labeling of renal dipeptidaseand easy detection. Similar inhibitors of other enzymes are also knownin the art and can be used. Inhibitors can be labeled using anydetectable moiety known in the art, including but not limited to fluorsand radioactive atoms.

[0035] RNA for any of the markers can be detected using any of the knowntechniques in the art. Preferably an amplification step will be used,because the amount of RNA for the marker is expected to be very smallfrom the sources contemplated. Suitable techniques include RT-PCR,hybridization of copy mRNA (cRNA) to an array of nucleic acid probes,and Northern blotting.

[0036] Protein forms of the markers can be detected using any techniquesknown in the art. These include activity assays, immunological assays,binding to specific ligands, etc. Particularly suitable assays for RenalDipeptidase include using L-L amino acid dipeptide substrates and L-Damino acid dipeptide substrates. Substrates which can be used forassaying renal dipeptidase are shown in FIG. 8, and include the genericstructures for dipeptides and dehydrodipeptides. ε (DNP)-L-Lysine-D-Ampcan also be used as a substrate, yielding a colored product. Substratesfor other enzymes can be used similarly to assess the presence of thetumor marker enzyme in the body or in a body sample. Such substrates canbe labeled with detectable moieties, including but not limited to fluorsand radioactive atoms. One particularly useful labeling scheme employs asubstrate which is labeled with two moieties on opposite sides of thesubstrate cleavage site. One of the moieties is fluorescent and one ofthe moieties is a quencher. When the two moieties are close, as in anintact substrate, the fluorescence of the fluorescent moiety isquenched. Upon cleavage the quenching is released and an increase influorescence is observed.

[0037] As mentioned above, inhibitors can also be labeled and used fordetecting suitable markers. In addition, antibodies can be used to labelprotein forms of the markers. The antibodies can be labeled as is knownin the art. Suitable radioactive atoms for use in labeling inhibitors,substrates, and antibodies include In-111, I-123, Tc-99m, Re-186,Re-188, Ga-67, Ga-68, Tl-201, Fe-52, Pb-203, Co-58, Cu-64, I-124, I-125,I-131, At-210, Br-76, Br-77, and F-18 and others known in the art forsuch purposes. Contrast enhancement agents can also be attached to thesubstrates, inhibitors, or antibodies. Such agents include gadolinium.Moreover, imaging techniques can be used to detect such labels withinthe body. An example of an imaging technique which can be used is spiralcomputer tomography. For this technique, the detecting agent, such asinhibitor or antibody can be linked to a contrast enhancing agent. Otherdetection means that can be used include gamma cameras, magneticresonance -imaging, planar scintigraphic imaging, SPECT imaging, PETimaging, and ultrasound imaging. Thus markers can be detected both insitu in the body or in vitro in an isolated body sample.

[0038] Epithelial cells can be isolated from blood or other tissuesamples to enrich for the markers or their mRNAs. Epithelial cells canbe isolated, inter alia, by immunoaffinity techniques. Such a techniqueis described in more detail below.

[0039] Substrates of enzymic markers can be administered to subjects andthe reaction products measured in body samples. Inhibitors can beadministered to subjects and the subject can be imaged to detect theinhibitor bound to the marker. Such markers are preferably those whichare not secreted proteins, but rather are those which are anchored to atumor. Typical modes of administration of such agents can be any whichis suitable, including but not limited to per os, intravenous,intramuscular, intraarterial, subdermal, transdermal, and rectal.

[0040] A high background of certain markers may obscure detection ofincreased expression. In such a situation, one can use tumor-specificglycoforms as a means of distinguishing between the background markerand the marker that is due to the tumor. Tumor-specific glycoforms ofRenal Dipeptidase and MIC-1 bind to LPHA, an L lectin from Phaseolusvulgaris hemagglutinin, and thus can be distinguished on that basis.Other lectins such as with similar specificity for tumor-specificglycoforms, such as Sambucus Nigra Lectin isolated from Sambucus nigra(elderberry) bark can be used as well.

[0041] Normal subjects are used as a comparison to the test subjects todetermine whether the amounts of markers observed in the feces or bloodare elevated. Preferably the normal subjects have been confirmed astumor-free by colonoscopy. More preferably several samples are pooled ortheir individual values are averaged to arrive at a normal value.

[0042] Some of the most highly overexpressed genes found in colorectaladenomas and colorectal cancers are discussed below. Regenerating IsletDerived Pancreatic Stone Protein, encoded by the REGA gene, is asecreted polypeptide first found in pancreatic precipitates and stonesfrom patients suffering from chronic pancreatitis (7). The cDNA encodingthis protein was isolated from a random screen of genes highly expressedin a regenerating-islet derived cDNA library (8) and subsequently shownto be elevated in colorectal cancers (9). More recently, REGA wasisolated in a hybridization-based screen for genes elevated incolorectal cancers and shown to be elevated in many colorectaladenocarcinomas (10). Consistent with these published observations, weobserved a strong elevation in expression of REGA in unpurified tumors,and a similar elevation in one purified tumor. In situ hybridizationexperiments demonstrated REGA to be strongly expressed in the epithelialcells of the tumors, with no expression evident in the stroma (FIG. 5A).

[0043] TGFB-induced gene (TGFBI) encodes a small polypeptide of unknownfunction initially isolated through a differential display screen forgenes induced in response to treatment with TGF β (11). The protein isexpressed in the keratinocytes of the cornea (12) and, interestingly,germline mutations of this gene cause familial corneal dystrophies (13).TGFBI was previously shown to be among the most significantly elevatedgenes in colorectal cancers (4), and our new data show that it isexpressed at high levels in adenomas as well. Quantitative PCR resultsdemonstrated strong elevation both in unpurified tumors and purifiedtumor epithelial cells. Accordingly, in situ hybridization experimentsrevealed TGFBI to be expressed in many cell types, in both the stromaland epithelial compartments (FIG. 5B).

[0044] Lysozyme (LYS, 1,4-β-N-acetylmuramidase, EC 3.2.1.17) is anenzyme with bacteriolytic activity (14) capable of cleaving β-1,4glycosidic bonds found in the cell walls of gram-positive bacteria. Theenzyme is expressed in the secretory granules of monocytes, macrophagesand leukocytes, as well as in the Paneth cells of the gastrointestinaltract. Fecal lysozyme levels are dramatically elevated in patients withinflammatory bowel disease (15, 16), and serum lysozyme activity issignificantly elevated in patients with sarcoidosis (17), both of whichare diseases characterized by aberrant chronic inflammation.Furthermore, lysozyme immunoreactivity has been observed in theepithelial cells of both adenomas and carcinomas of the large intestine(18). In our study, the degree of elevation of expression of LYS variedfrom 4-fold to 55-fold in the unpurified samples. In contrast, thedegree of elevation of expression of LYS observed in purified epithelialcells was only 2-5 fold. This suggested that a substantial portion ofthe expression for this gene in the tumors could have been derived fromnon-epithelial cells. Consistent with this hypothesis, in situhybridization experiments revealed that the majority of LYS mRNA waspresent in a stromal component that appeared to be macrophages (FIG.5C). The expression of LYS in the macrophage compartment of colorectaltumors was also supported by its high representation in a SAGE libraryconstructed from hematopoietic cells (CD45+, CD64+, CD14+) purified fromcolorectal tumors (602 LYS tags/56,643 total tags) (6).

[0045] One interesting gene identified in the current study is renaldipeptidase (RDP). RDP is a GPI-anchored enzyme whose major site ofexpression is the epithelial cells of the proximal tubules of the kidney(reviewed in (19)). The enzyme has been extensively analyzed withrespect to its catalytic mechanism and inhibition kinetics by a varietyof synthetic inhibitors. RDP is unique among the dipeptidases in that itcan cleave amide bonds in which the C-terminal partner is a D aminoacid, providing excellent opportunity for the development of specificprobes for its detection in vivo. Quantitative PCR revealed RDP to bemarkedly elevated in both unpurified and purified tumor epithelialcells, and in situ hybridization experiments showed that RDP wasexclusively localized to epithelial cells of colorectal tumors (FIG.5D).

[0046] Macrophage Inhibitory Cytokine (MIC-1) is a small polypeptide of16 kDa first isolated from a differential screen for genes that wereinduced upon macrophage activation (20). Concurrently, it was identifiedin the IMAGE database by a search for molecules homologous to the BoneMorphogenic Protein/TGF β family of growth and differentiation factors(21). In addition to being highly expressed in activated macrophages,MIC-1 has been noted to be highly expressed in placenta and theepithelial cells of normal prostate. In the current study, we foundMIC-1 expression to be elevated between 7 and 133 fold in the unpurifiedtumors. As observed for LYS, the purified tumor cells demonstratedsignificant but less elevation of expression of MIC-1 (5 to 7-fold)indirectly implicating stromal expression to be partly responsible forthe dramatic elevation seen in some tumors. Consistent with thishypothesis, in situ hybridization experiments revealed expression inboth the epithelium of the tumor, and in a cell type resemblinginfiltrating macrophages (FIG. 5E).

EXAMPLES Example1 SAGE

[0047] In an effort to identify potential molecular markers of earlycolorectal tumors, we have here analyzed gene expression in benign andmalignant colorectal tumors in an unbiased and comprehensive fashion. Weused SAGE to analyze global gene expression in normal, benign andmalignant colorectal tissue. SAGE is a gene expression profiling methodthat associates individual mRNA transcripts with 15-base tags derivedfrom specific positions near their 3′ termini (3). The abundance of eachtag provides a quantitative measure of the transcript level presentwithin the mRNA population studied. SAGE is not dependent onpre-existing databases of expressed genes, and therefore provides anunbiased view of gene expression profiles. For the current study, SAGElibraries derived from two samples of normal colonic epithelium, twocolorectal adenomas, and two colorectal cancers were analyzed. Theselibraries contained a combined total of 290,394 transcript tagsrepresenting 21,343 different transcripts (Table 1). TABLES 1 Summary ofSAGE data Total number Number of of tags different transcripts SAGELibrary observed observed* Normal Colorectal Epithelium NC-1  49,6109,359 NC-2  48,479 9,610 Adenomas Ad-A  52,573 11,167 Ad-B  42,661 9,483Cancers Tu-98  41,371 9,780 Tu-102  55,700 11,039 Total 290,394 21,343

[0048] Two comparisons were performed, one between the adenoma andnormal samples, and one between the cancer and normal samples. Thesecomparisons revealed 957 transcript tags that were differentiallyexpressed more than 2-fold between normal and tumor tissue (Table 2). Acomparison of the fold change in adenomas versus cancers revealed thatmany transcripts were similarly elevated or repressed in both adenomasand cancers although the magnitude often varied (FIG. 1A). Indeed themajority (79%) of comparisons were in quadrants of the plot indicativeof concordant elevation. TABLE 2 Differentially expressed transcripts inbenign and malignant tumor colorectal tissue Elevated in both Repressedin Total transcripts Fold change in Elevated in Elevated in adenomas andRepressed in Repressed in both adenomas differentially expressionadenomas^(a) cancers^(a) cancer^(a) adenomas^(b) cancers^(b) andcancers^(b) expressed 2 346 170 50 313 380 192 957 4 263 119 23 225 270117 735 10 160 79 10 134 157 58 462 20 49 40 9 72 52 23 181

[0049] From both practical and biological perspectives, those changesshowing the greatest magnitude were deemed the most interesting. In thisregard, 49 tags were identified to be elevated by ≧20-fold in theadenomas and 40 were elevated by ≧20-fold in the cancers (Table 2).Conversely, there were 72 transcripts that were decreased by ≧20-fold inadenomas and 52 decreased by ≧20-fold in the cancers (Table 2).

[0050] There were nine transcripts that were elevated by ≧20-fold inboth adenomas and cancers (FIG. 1B and Table 3) and 23 that wererepressed by ≧20-fold (Table 4). We were especially interested in geneswhose products were predicted to be secreted or displayed on the cellsurface, as these would be particularly suitable for the development ofserologic or imaging tests for presymptomatic neoplasia, respectively.We were able to identify six such genes (TGFBI, LYS, RDP, MIC-1, REGAand DEHL) from among those whose transcript tags were elevated in bothadenoma and carcinoma SAGE libraries. TABLE 3 Transcripts most elevatedin adenomas and cancers^(a) Normal Adenomas Cancers Tag Sequence NC-1NC-2 AD-A1 AD-B2 Tu-98 Tu-102 Transcript name ATGTAAAAAA 0 0 26 32 2 12Lysozyme (LYS) TAATTTTTGC 0 1 99 12 20 37 Differentially Expressed inHematopoietic Lineages (DEHL) GTGTGTTTGT 0 0 17 29 17 15 TransformingGrowth Factor, Beta-Induced (TGFBI) GTGCTCATTC 0 0 13 7 2 10 MacrophageInhibitory Cytokine, 1 (MIC-1) TTCCAGCTGC 0 0 7 6 2 9 Adaptor-relatedProtein Complex 2, alpha 2 subunit^(b) ACCATTGGAT 0 0 3 10 3 9Interferon Induced Transmembrane Protein 1 (9-27)^(b) TTTCCACTAA 0 0 8 44 6 Regenerating Islet-Derived 1 alpha (REGA) CAAGGACCAG 0 0 5 6 10 12Renal Dipeptidase (RDP) AGGACCATCG 0 0 8 2 1 18 Defensin, Alpha 5,Paneth cell-specific^(c)

[0051] TABLE 4 Transcripts most repressed in adenomas and cancers^(a)Normal Adenoma Cancer Tag Sequence NC-1 NC2 AD-A1 AD-B Tu-981 Tu-102 UNIID Transcript name GTCATCACCA 35 22 0 0 0 0 32966 Guanylate CyclaseActivator 2B CCTTCAAATC 29 17 0 0 1 0 23118 Carbonic Anhydrase ITCTGAATTAT 24 16 0 0 1 0 50964 Carcinoembryonic Antigen-Related CellAdhesion Molecule 1 TTATGGTGTG 11 17 0 0 0 0 271499 ESTs CTGGCAAAGG 1422 1 0 0 0 72789 hypothetical protein FLJ20217 AGGTGACTGG 10 14 0 0 0 0No Match CTTATGGTCC 36 11 0 1 1 0 179608 Retinol Dehydrogenase HomologATGATGGCAC 12 32 1 0 1 0 84072 Transmembrane 4 Superfamily Member 3GTCCGAGTGC 17 3 0 0 0 0 3337 Transmembrane 4 Superfamily Member 1ATTTCAAGAT 35 21 0 2 1 0 155097 Carbonic Anhydrase II (CA2) CAAGAGTTTC14 2 0 0 0 0 183617 ESTs GCCATCCTCC 9 13 0 1 0 0 No Match ACCCAACTGC 123 0 0 0 0 232604 Homo sapiens cDNA: FLJ22675 fis, clone HSI10553GCCCACGTCA 7 8 0 0 0 0 No Match TTTGGTTTCA 2 13 0 0 0 0 No MatchCTCAGAACTT 18 3 1 0 0 0 194710 N-acetylglucosaminyl transferase 3, mucintype CCAACACCAG 9 19 1 0 0 1 181165 Eukaryotic Translation ElongationFactor 1 Alpha 1 GCCACATACT 3 9 0 0 0 0 4984 KIAA0828 protein GTATTGGGGC5 7 0 0 0 0 No Match CCGGCTTGAG 7 4 0 0 0 0 2722 Inositol1,4,5-trisphosphate 3-Kinase A GATATGTAAA 1 10 0 0 0 0 227059 ChlorideChannel, Calcium Activated, Family Member 4 CATAGGTTTA 66 39 4 1 5 01650 Solute Carrier Family 26, member 3 (DRA) GTCCTGAACA 7 3 0 0 0 078546 ATPase, Ca++ Transporting, Plasma Membrane 1

[0052] SAGE. For the initial SAGE³ of benign tumors, fresh adenomas wereobtained from surgical specimens derived from FAP patients. Adenomasfrom FAP patients were employed because of the ready availability ofsmall lesions and the certainty of inactivation of the APC pathway whichinitiates the formation of the majority of sporadic tumors. Afterhistopathological verification of the neoplastic nature of the lesion(>70% neoplastic cells), total RNA was isolated by solubilizing thetissue in RNAgents Lysis Buffer (Promega, Madison, Wis.) followed byultracentrifugation over a cesium chloride gradient. mRNA selection wasperformed from the purified total RNA using oligo(dT) cellulose (LifeTechnologies, Gaithersburg, Md.). Two adenoma SAGE libraries wereprepared as described (3, 4) and sequenced to a total depth of over90,000 transcript tags. For SAGE of normal and malignant tissues, fourpreviously described normal (NC-1 and NC-2) and primary cancer (Tu-98and Tu-102) SAGE libraries were employed (4). In collaboration with theCancer Genome Anatomy Project (CGAP) (5), the analyses of theselibraries was extended from a total of 123,046 transcripts in thepreviously published work to 195,160 transcripts in the current work.Tags were extracted from the raw sequence data and, after excludingrepeated ditags, linker sequences, and tags from the polyrnorphic MajorHistocompatibility loci, the resulting tag libraries were compared andstatistical analysis performed using SAGE software, version 4.0. Datafrom the libraries are publicly available at the Uniform ResourceLocator (URL) address for the http file type found on the www hostserver that has a domain name of ncbi.nlm.nih.gov, and a path to thedirectory SAGE, and detailed SAGE protocols are available at the UniformResource Locator (URL) address for the http file type found on the wwwhost server that has a domain name of sagenet.org, and file name ofsage_protocol.htn.

Example 2 RT-PCR

[0053] To verify the increased expression of these six genes, we usedquantitative RT-PCR techniques to analyze the expression in sevencolorectal neoplasms (three sporadic adenomas and four sporadic cancers)and matched normal colonic mucosa. For these assays, specific primerswere developed that resulted in amplification from cDNA but not genomicDNA. Controls were provided by similar quantitative PCR assays of a genewhose expression was found to be very similar in the SAGE libraries ofnormal and neoplastic colon (β-amyloid precursor protein). Thequantitative PCR experiments verified that five of the six selectedgenes (TGFBI, LYS, RDP, MIC-1, REGA) were expressed at significantlyhigher levels in every neoplastic sample analyzed compared topatient-matched normal mucosa (FIG. 2). Several tumors exhibited≧20-fold higher levels of the studied transcripts compared to theirpatient-matched normal colonic mucosa, as predicted by SAGE. Anothercontrol was provided by the quantitative PCR analysis of four geneswhose expression was observed to be reduced in the SAGE librariesprepared from adenomas and cancers compared to those from normal colonicmucosa. As shown in FIG. 3, the quantitative PCR confirmed the lowerlevels of expression of each of these genes, emphasizing that thedramatic elevations in expression observed in FIG. 2 representedgene-specific phenomena.

[0054] Quantitative PCR. Tumors were collected, snap frozen, and storedat -80° C. They were verified to be predominantly composed of neoplasticcells by histopathological analysis. mRNA was isolated from tumors andpatient-matched normal colonic mucosa using QuickPrep reagents (AmershamPharmacia Biotech UK, Buckinghamshire, England), and single-strandedcDNA was synthesized using Superscript II (Life Technologies,Gaithersburg, Md.). Quantitative PCR was performed using an iCycler(Bio-Rad, Hercules, Calif.), and threshold cycle numbers determinedusing iCycler software, version 2.1. Reactions were performed intriplicate and threshold cycle numbers averaged. All genes examined werenormalized to a control gene (□-amyloid precursor protein, shown by SAGEto be expressed at equivalent levels in all colorectal samples), andfold induction calculated according to the formula2^((Rt-Et))/2^((Rn-En)) where Rt is the threshold cycle number for theReference gene observed in the tumor, Et is the threshold cycle numberfor the Experimental gene observed in the tumor, Rn is the thresholdcycle number for the Reference gene observed in the normal, and En isthe threshold cycle number for the Experimental gene observed in thenormal. The primers used for quantitative PCR were obtained fromGeneLink (Hawthorne, N.Y.), and their sequences are available uponrequest.

Example 3 Expression in Isolated Epithelial Cells

[0055] The quantitative PCR data obtained from mRNA isolated from wholetumors provided independent evidence that SAGE provided an accurateindication of gene expression changes in colorectal neoplasia. However,neither analysis identified the cell types responsible for the increasedexpression. Non-neoplastic stromal cells within tumors may beconsiderably different than those in normal colonic mucosa (6), and theepithelial derivation of gene expression differences cannot reliably beconcluded without direct supporting evidence. We therefore sought todetermine if the epithelial cells of cancers express elevated levels ofthe six genes depicted in FIG. 2. First, we affinity-purified cancerousand patient-matched normal epithelial cells from fresh surgicalspecimens using immunomagnetic beads directed to the pan epithelialmarker Ber-EP4, prepared cDNA and performed quantitative PCR analysis todetermine the expression levels of the elevated genes as above. Elevatedexpression was observed in the purified tumor epithelial cells for eachof the six genes examined (FIG. 4), demonstrating that at least some ofthe increased expression was derived from epithelial cells. However,relative expression of LYS was not as prominent or reproducible in thepurified epithelial cells as in the mRNA from the unfractionated tumors,suggesting that other cell types might have contributed transcripts fromthis gene.

[0056] Epithelial cell immunoaffinity purification. Tumor epithelialcells were purified using a modification of the procedure previouslydeveloped for the isolation of tumor endothelial cells (6). In brief,fresh surgical specimens of tumor and matched normal tissue wereobtained and digested with collagenase and the resulting materialfiltered through a nylon mesh to obtain single cell suspensions. Thecells were then bound to a mixture of anti-CD14 and anti-CD45immunomagnetic beads (Dynal, Oslo, Norway) to deplete the population ofhematopoetic cells (negative selection). The remaining cell suspensionwas then incubated with anti-Ber-EP4 immunomagnetic beads to isolateepithelial cells (positive selection). Purified cells were lyseddirectly on the beads and mRNA purified using QuickPrep reagents(Amersham Pharmacia Biotech UK, Buckinghamshire, England).

Example 4 In situ Hybridization in Multiple Tumors

[0057] We performed in situ hybridization to RNA in frozen sections oftumors for five of the genes showing the most consistent elevation. DEHLwas found to be elevated in only five of the nine tumors examined andwas not investigated further. To increase the sensitivity of detection,we generated several RNA probes for each tested gene using in vitrotranscription techniques. The results obtained are discussed below inconjunction with brief overviews of each of the five genes of interest.

[0058] In situ Hybridization. Non-radioactive in situ hybridization wasperformed as described (6). For each gene analyzed, a cocktail ofanti-sense probes made through in vitro transcription were employed toincrease sensitivity. The primers used to generate templates for thesynthesis of the in situ riboprobes were obtained from GeneLink(Hawthorne, N.Y.), and their sequences are available upon request.

[0059] The results summarized above show that although a large number oftags are observed in the colorectal tissues analyzed, only a smallfraction (957/21,343, <5%) were expressed differentially in benign ormalignant neoplastic tissues. A similarly small fraction of genes(66/4000, 1.7%) were found to be aberrantly expressed in colorectalneoplasms using oligonucleotide arrays (22). Analysis of thesedifferentially expressed genes not only has the potential to provideinsights into the biology of human neoplasia but also may haveclinically useful applications. One of the most exciting potentialapplications concerns the identification of genes whose products providecellular and serum markers for colorectal neoplasia. In the currentstudy, we identified several genes that appeared to meet all of thesecriteria and may therefore be especially useful as diagnostic tools forthe early detection of presymptomatic colorectal neoplasia. Indeed, theproduct of one of these genes (MIC-1), has recently been found to beelevated in the serum of patients with colorectal and other cancers,providing further validation of this approach (24).

[0060] While the invention has been described with respect to specificexamples including presently preferred modes of carrying out theinvention, those skilled in the art will appreciate that there arenumerous variations and permutations of the above described systems andtechniques that fall within the spirit and scope of the invention as setforth in the appended claims. TABLE 5 Tag_Sequence NC1 NC2 AD1 AD2 CA1CA2 UNI ID Description AAAAGAAACT 1 3 33 52 7 16 172182 poly(A)-bindingprotein, cytoplasmic 1 AACGAGGAAT 8 0 24 26 17 23 AAGAAGATAG 6 6 28 2521 34 184776 ribosomal protein L23a AATAGGTCCA 12 9 32 36 22 22 113029ribosomal protein S25 ACAACTCAAT 1 1 7 7 8 6 244125 EST ACAACTCAAT 1 1 77 8 6 75922 brain protein 13 ACATCATCGA 10 18 50 66 34 46 182979ribosomal protein L12 ACCATTGGAT 0 0 3 10 3 9 146360 interferon inducedtransmembrane protein 1 (9-27) ACCTGTATCC 5 3 20 6 26 35 182241interferon induced transmembrane protein 3 (1-80) ACTCCAAAAA 9 12 21 6521 37 133230 ribosomal protein S15 AGCACCTCCA 37 37 108 81 57 108 75309eukaryotic translation elongation factor 2 AGGACCATCG 0 0 8 2 1 18AGGGCTTCCA 26 41 74 108 50 85 29797 ribosomal protein L10 ATGGCTGGTA 1846 79 75 81 136 182426 ribosomal protein S2 ATGTAAAAAA 0 0 26 32 2 12178112 DNA segment, single copy probe LNS-CAI/LNS-CAII (deleted inpolyposis ATGTAAAAAA 0 0 26 32 2 12 234734 lysozyme (renal amyloidosis)ATGTAAAAAA 0 0 26 32 2 12 83715 Sjogren syndrome antigen B (autoantigenLa) ATTCTCCAGT 8 20 20 48 43 28 234518 ribosomal protein L23 CAAGGACCAG0 0 5 6 10 12 109 dipeptidase 1 (renal) CAATAAATGT 8 6 40 76 33 67179779 ribosomal protein L37 CAGCTCACTG 4 17 9 35 21 24 158675 ribosomalprotein L14 CATTTGTAAT 48 27 102 57 36 125 CCTAGCTGGA 16 27 58 45 48 66182937 peptidylprolyl isomerase A (cyclophilin A) CCTTCGAGAT 6 12 13 297 41 76194 ribosomal protein S5 CTCCTCACCT 7 13 38 36 24 75 242908lecithin-cholesterol acyltransferase CTGACTTGTG 0 0 1 20 9 2 77961 majorhistocompatibility complex, class I, B CTGGGTTAAT 14 24 84 83 42 112126701 ribosomal protein S19 CTGTTGATTG 13 3 60 38 32 27 249495heterogeneous nuclear ribonucleoprotein A1 CTGTTGGTGA 9 19 37 59 31 613463 ribosomal protein S23 GAAAAATGGT 7 12 49 47 25 27 181357 lamininreceptor 1 (67 kD, ribosomal protein SA) GAGTCAGGAG 2 0 8 6 9 7 181271CGI-120 protein GCATAATAGG 11 16 22 54 50 21 184108 ribosomal proteinL21 (gene or pseudogene) GCATTTAAAT 1 2 10 18 12 7 261802 eukaryotictranslation elongation factor 1 beta 1 GCATTTAAAT 1 2 10 18 12 7 275959eukaryotic translation elongation factor 1 beta 2 GCATTTGACA 2 5 27 17 920 172129 Homo sapiens cDNA: FLJ21409 fis, clone COL03924 GCTTTTAAGG 2 814 32 16 17 8102 ribosomal protein S20 GGACCACTGA 18 39 76 57 48 83119598 ribosomal protein L3 GGGGGTAACT 1 2 8 11 14 13 99969 fusion,derived from t(12;16) malignant liposarcoma GTGCGCTGAG 0 0 75 0 20 18277477 major histocompatibility complex, class I, C GTGCTCATTC 0 0 13 72 10 116577 prostate differentiation factor GTGCTCATTC 0 0 13 7 2 1025945 ESTs GTGTGTTTGT 0 0 17 29 17 15 118787 transforming growth factor,beta-induced, 68 kD GTTCGTGCCA 1 13 18 43 24 18 179606 nuclear RNAhelicase, DECD variant of DEAD box family GTTCGTGCCA 1 13 18 43 24 18179666 uncharacterized hypothalamus protein HSMNP1 TAATAAAGGT 4 11 37 6224 27 151604 ribosomal protein S8 TAATTTTTGC 0 1 99 12 20 37 273321differentially expressed in hematopoietic lineages TCACAAGCAA 10 7 17 2113 38 146763 nascent-polypeptide-associated complex alpha polypeptideTCAGATCTTT 14 32 37 108 31 87 75344 ribosomal protein S4, X-linkedTCCTGCCCCA 1 5 10 14 7 16 171814 parathymosin TGAAATAAAA 0 2 2 14 13 11173205 nucleophosmin (nucleolar phosphoprotein B23, numatrin) TGAAATAAAA0 2 2 14 13 11 192822 Human DNA sequence from clone RP5-1179L24 onchromosome 6q24.3-25.3. Contains the 3′ end of the gene for a novelprotein similar to mouse phospholipase C neighboring protein PNG, ESTs,STSs and GSSs TGATGTCTGG 0 0 2 6 8 2 83883 transmembrane, prostateandrogen induced RNA TGTAATCAAT 2 3 13 11 8 11 249495 heterogeneousnuclear ribonucleoprotein A1 TTACCATATC 10 5 22 30 26 22 300141ribosomal protein L39. TTATGGGATC 6 4 24 37 36 47 5662 guaninenucleotide binding protein (G protein), beta polypeptide 2-like 1TTCAATAAAA 8 14 79 111 36 50 177592 ribosomal protein, large, P1TTCCAGCTGC 0 0 7 6 2 9 112442 ESTs, Weakly similar to TTCCAGCTGC 0 0 7 62 9 19121 adaptor-related protein complex 2, alpha 2 subunit TTCCAGCTGC0 0 7 6 2 9 227277 sine oculis homeobox (Drosophila) homolog 3TTTCCACTAA 0 0 8 4 4 6 1032 regenerating isler-derived 1 alpha(pancreatic stone protein, pancreatic thread protein) TTTCCACTAA 0 0 8 44 6 289088 heat shock 90 kD protein 1, alpha TTTTTAATGT 0 2 12 13 6 7161307 H3 histone, family SA

[0061] TABLE 6 Tag_Sequence NC1 NC2 AD1 AD2 CA1 CA2 UNI ID DescriptionAAATCTGGCA 16 15 2 4 2 0 430 plastin 1 (I isoform) AACGTGCAGG 29 31 13 67 8 160786 argininosuccinate synthetase AAGAAAGCTC 20 6 0 2 1 5 25264DKFZP434N126 protein AAGAAAGCTC 20 6 0 2 1 5 91011 anterior gradient 2(Xenepus laevis) homolog AAGAAGCAGG 8 16 3 4 1 3 11441 chromosome 1 openreading frame 8 AAGGTAGCAG 15 16 2 4 2 4 104125 adenylylcyclase-associated protein AATAAAGGCT 25 11 3 7 3 4 179735 ras homologgene family, member C AATAGTTTCC 7 16 2 3 6 1 272620 pregnancy specificbeta-1-glycoprotein 9 AATCACAAAT 18 45 1 4 14 3 74466 carcinoembryonicantigen-related cell adhesion molecule 7 AATGAGAAGG 11 3 0 0 1 0 198248UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1ACAATTGGTC 0 10 0 0 0 0 155097 carbonic anhydrase II ACACCCATCA 2 27 1 05 3 110445 CGI-97 protein ACAGGGTGAC 25 13 1 8 7 6 174050 endothelialdifferentiation-related factor 1 ACATTGGGTG 377 334 67 34 96 33 275086PR domain containing 10 ACATTGGGTG 377 334 67 34 96 33 5241 fatty acidbinding protein 1, liver ACCCAACTGC 12 3 0 0 0 0 232604 Homo sapienscDNA: FLJ22675 fis, clone HSI10553 ACCCACGTCA 22 10 3 5 3 1 198951 jun Bproto-oncogene ACCCCCCCGC 44 38 7 13 16 6 229413 ESTs ACCCCCCCGC 44 38 713 16 6 2780 jun D proto-oncogene ACCTGCATCC 0 12 0 0 0 0 ACCTGGGGAG 3511 1 3 4 3 131748 ESTs, Moderately similar to ACCTGGGGAG 35 11 1 3 4 3209119 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidicacid acyltransferase, beta) ACGGTCCAGG 5 12 0 0 0 1 72924 cytidinedeaminase ACTCTTGTTG 7 2 0 0 0 0 5378 spondin 1, (f-spondin)extracellular matrix protein ACTGTGGCGG 17 34 9 12 9 4 112242 ESTsAGAATAGCTT 44 67 3 9 11 30 24133 ESTs AGCAGGAGCA 50 14 6 3 7 6 178292KIAA0180 protein AGCAGGAGCA 50 14 6 3 7 6 738 early growth response 1AGCCCGACCA 16 8 2 4 1 3 104114 H. sapiens HCG I mRNA AGGATGGTCC 34 19 53 6 8 71779 Homo sapiens DNA from chromosome 19, cosmld F21856AGGCCAAGGG 21 6 3 1 3 4 76057 galactose-4-epimerase, UDP AGGTGACTGG 1014 0 0 0 0 AGTGGGCTCA 3 8 0 1 0 0 ATACTCCACT 82 59 0 0 10 3 778guanylate cyclase activator 1B (retina) ATATAATCTG 18 14 1 4 5 5 621lectin, galactoside-binding, soluble, 3 (galectin 3) ATCGTGGCGG 193 9219 25 20 10 5372 claudin 4 ATGACGCTCA 22 18 6 5 3 4 8254 hypotheticalprotein PRO0899 ATGATGGCAC 12 32 1 0 1 0 84072 transmembrane 4superfamily member 3 ATGCGGAGTC 14 13 5 3 2 4 25527 tight junctionprotein 3 (zona occludens 3) ATGCGGGAGA 38 39 15 22 5 6 109748 Homosapiens CAC-1 mRNA, partial cds ATGGCACGGA 6 21 1 1 3 0 81097 cytochromeC oxidase subunit VIII ATGGTCTACG 10 5 0 0 1 0 96593 hypotheticalprotein ATGGTGGGGG 25 30 11 9 1 2 1665 zinc finger protein homologous toZfp-36 in mouse ATGTGCGTGG 38 8 11 3 7 7 56937 suppression oftumorigenicity 14 (colon carcinoma, matriptase, epithin) ATGTGGGCTC 7 20 0 0 0 151641 glycoprotein A repetitions predominant ATGTGGGCTC 7 2 0 00 0 27018 Ris ATTGGAGTGC 136 85 15 36 37 19 220529 carcinoembryonicantigen-related cell adhesion molecule 5 ATTTCAAGAT 35 21 0 2 1 0 155097carbonic anhydrase II ATTTCAAGAT 35 21 0 2 1 0 24453 ESTs CAAATAAAAG 912 2 0 1 1 185055 BENE protein CAAGAGTTTC 14 2 0 0 0 0 183617 ESTsCACCCCTGAT 73 169 58 58 13 36 173724 creatine kinase, brain CAGTGCGTTC12 3 0 0 3 0 8302 four and a half LIM domains 2 CATAGGTTTA 66 39 4 1 5 01650 solute carrier family 26, member 3 CCAAAGCTAT 42 16 14 12 6 1184072 transmembrane 4 superfamily member 3 CCAACACCAG 9 19 1 0 0 1181165 eukaryotic translation elongation factor 1 alpha 1 CCACTGCACC 2119 5 5 6 14 CCAGGGGAGA 45 66 24 22 10 20 278613 interferon,alpha-inducible protein 27 CCATTCCACT 13 1 2 0 0 0 CCCAACGCGC 106 1 3 50 2 272572 hemoglobin, alpha 2 CCCCCGAAGC 25 27 3 15 4 8 61265 ESTs,Weakly similar to CCCCCGCGGA 33 25 6 11 4 12 95697 liver-specificbHLH-Zip transcription factor CCCCCTGCAT 5 4 0 0 0 0 CCCGCCTCTT 0 40 3 316 1 mito Tag matches mitochondrial sequence CCCTCCCGAA 89 54 9 14 18 75940 hypothetical protein FLJ20063 CCGCTCCACT 127 102 55 46 37 30CCGGCTTGAG 7 4 0 0 0 0 2722 inositol 1,4,5-trisphosphate 3-kinase ACCTCCAGCTA 715 458 142 125 131 147 242463 keratin 8 CCTCCAGTAC 20 8 2 32 4 CCTGCCCCCC 20 30 6 3 11 4 861 mitogen-activated protein kinase 3CCTGCTGCAG 7 34 0 1 6 9 102482 mucin 5, subtype B, tracheobronchialCCTGCTTGTC 20 23 0 3 0 5 268171 ESTs, Weakly similar to CCTGCTTGTC 20 230 3 0 5 2719 epididymis-specific, whey-acidic protein type,four-disulfide core; putative ovarian carcinoma marker CCTGGAAGAG 30 2611 4 12 16 75655 procollagen-proline, 2-oxoglutarate 4-dioxygenase(proline 4-hydroxylase), beta polypeptide (protein disulfide isomerase;thyroid hormone binding protein p55) CCTGTCTGCC 14 22 0 1 1 1 107139hypothetical protein CCTGTGACAG 22 27 0 4 3 1 120 anti-oxidant protein 2(non-selenium glutathlone peroxidase, acidic calcium- independentphospholipase A2) CCTTCAAATC 29 17 0 0 1 0 23118 carbonic anhydrase ICGAGGGGCCA 110 47 18 18 12 32 182485 actinin, alpha 4 CGCTGTGGGG 58 5319 8 8 6 7486 protein expressed in thyroid CGGACTCACT 20 45 5 14 14 10284134 serologically defined colon cancer antigen 28 CGGACTCACT 20 45 514 14 10 84700 similar to phosphatidylcholine transfer protein 2CGGGAGTCGG 28 30 13 2 9 1 236720 ESTs, Weakly similar to CGGTGGGACC 7 141 1 3 3 99175 Homo sapiens cDNA: FLJ21606 fis, clone COL07302 CGTGGGTGGG1 10 1 0 0 1 202833 heme oxygenase (decycling) 1 CTAGCCTCAC 172 90 30 5336 58 14376 actin, gamma 1 CTCAGAACTT 18 3 1 0 0 0 194710 glucosaminyl(N-acetyl) transferase 3, mucin type CTGAACCTCC 5 15 2 0 0 0 4205hypothetical protein FLJ20124 CTGACCTGTG 88 130 48 18 16 46 77961 majorhistocompatibility complex, class I, B CTGGATCTGG 21 21 3 9 5 10 75658phosphorylase, glycogen; brain CTGGCAAAGG 14 22 1 0 0 0 CTGGCCCTCG 18652 1 3 15 14 1406 trefoil factor 1 (breast cancer, estrogen-induciblesequence expressed in) CTGGCCCTCG 186 52 1 3 15 14 166184 Intersectin 2CTGCCCCTCG 186 52 1 3 15 14 7720 dynein, cytoplasmic, heavy polypeptide1 CTGGCTATCC 7 3 0 0 0 1 10784 hypothetical protein FLJ20037 CTGGGCCTCT22 22 2 2 3 3 50868 solute carrier family 22 (organic cationtransporter), member 1-like CTGTACTTGT 9 5 1 1 0 0 75678 FBJ murineosteosarcoma viral oncogene homolog B CTGTGTGGCT 0 12 1 0 0 0 127610acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain CTGTGTGGCT 0 12 10 0 0 54277 DNA segment on chromosome X (unique) 9928 expressed sequenceCTTACAAGCA 21 13 2 3 4 3 mito Tag matches mitochondrial sequenceCTTAGAGGGG 16 22 0 1 1 1 155191 vitlin 2 (ezrin) CTTATGGTCC 36 11 0 1 10 179608 retinol dehydrogenase homolog CTTCCAGCTA 64 31 22 20 14 19217493 annexin A2 CTTCTTGCCC 29 2 2 2 0 1 251577 hemoglobin, alpha 1CTTGACATAC 18 20 4 4 0 0 171695 dual specificity phosphatase 1CTTGATTCCC 26 9 5 0 2 5 77266 quiescin Q6 GACATCAAGT 198 87 23 14 47 17182265 keratin 19 GACCAGCCCA 23 21 3 2 12 5 75799 protease, serine, 8(prostasin) GACCAGTGGC 21 44 4 0 2 0 143131 glycoprotein A33(transmembrane) GACGCGGCGC 30 47 10 17 12 17 301684 RNA POLYMERASE I ANDTRANSCRlPT RELEASE FACTOR GAGAGCTCCC 5 11 3 0 2 1 mito Tag matchesmitochondrial sequence GAGCACCGTG 7 4 1 0 0 1 GATATGTAAA 1 10 0 0 0 0GATCCCAACT 9 29 5 7 1 1 118786 metallothionein 2A GATGAATCCG 12 14 2 2 12 283552 ESTs, Weakly similar to GATGACCCCC 42 49 4 3 3 3 mito Tagmatches mitochondrial sequence GCAAGAAAGT 48 0 0 4 0 1 155376hemoglobin, beta GCACAGGTCA 5 9 1 0 1 1 GCACCCTTTC 13 5 0 0 1 0GCACCTGTCG 2 9 0 0 1 0 109059 mitochondrial ribosomal protein L12GCACCTGTCG 2 9 0 0 1 0 1239 alanyl (membrane) aminopeptidase(aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13,p150) GCAGCTCCTG 13 47 3 2 7 3 119257 ems1 sequence (mammary tumor andsquamous cell carcinoma-associated (p80/85 src substrate) GCAGGAGGTG 213 0 0 0 1 11441 chromosome 1 open reading frame 8 GCAGGAGGTG 2 13 0 0 01 78040 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retentionreceptor 1 GCAGGGCCTC 128 165 41 69 39 51 92323 FXYD domain-containingIon transport regulator 3 GCCACATACT 3 9 0 0 0 0 4984 KIAA0828 proteinGCCACGTGGA 19 16 5 2 7 1 103665 villin-like GCCAGACACC 19 9 3 4 1 2 3804DKFZP564C1940 protein GCCAGGTTGC 14 5 1 1 1 1 42824 hypothetical proteinFLJ10718 GCCAGGTTGC 14 5 1 1 1 1 55682 eukaryotic translation initiationfactor 3, subunit 7 (zeta, 66/67 kD) GCCAGGTTGC 14 5 1 1 1 1 78996proliferating cell nuclear antigen GCCATCCTCC 9 13 0 1 0 0 GCCCACACAG 150 1 1 0 0 1690 heparin-binding growth factor binding protein GCCCACGTCA7 8 0 0 0 0 GCCCAGGGCC 4 44 2 1 2 1 10326 coatomer protein complex,subunit epsilon GCCCAGGGCC 4 44 2 1 2 1 229417 EST, Moderately similarto GCCCAGGGCC 4 44 2 1 2 1 229546 EST GCCCAGGTCA 519 447 136 128 58 22154903 ESTs, Weakly similar to GCCCAGTGGC 51 0 8 15 2 5 143131glycoprotein A33 (transmembrane) GCCGACCAGG 46 47 15 8 19 9 75741amiloride binding protein 1 (amine oxidase (copper-containing))GCCGGGTGGG 207 149 18 24 68 67 74631 basigin GCCGTGGAGA 32 23 4 11 7 780680 major vault protein GCCTGGCCAT 26 34 4 6 10 14 5662 guaninenucleotide binding protein (G protein), beta polypeptide 2-like 1GCCTGGCCAT 26 34 4 6 10 14 63042 DKFZp564J157 protein GCGAAACCCT 167 565123 43 64 98 GCGAAACTCG 5 9 1 0 0 0 GCGCAGAGGT 2 16 1 0 0 1 108124ribosomal protein L41 GCTCTTCCCC 9 21 1 2 2 0 33455 peptidyl argininedeiminase, type II GCTGCCCTTG 44 6 13 6 6 18 272897 Tubulin, alpha,brain-specific GCTGCCCTTG 44 6 13 6 6 18 278242 tubulin, alpha,ubiquitous GCTGGCACAT 15 14 1 0 6 0 179704 meprin A, alpha (PABA peptidehydrolase) GCTGGCCCCG 5 11 0 0 0 1 8185 CGI-44 protein; sulfidedehydrogenase like (yeast) GCTGTGCCTG 36 42 2 4 11 8 58247 protease,serine, 4 (trypsin 4, brain) GCTTGGGGAT 11 8 2 0 0 0 5394 myosin, heavypolypeptide-like (110 kD) GGAACAGGGG 1 13 1 0 0 2 102336 Rho GTPaseactivating protein 8 GGAACAGGGG 1 13 1 0 0 2 272972 hypothetical proteinFLJ20185 GGAACAGGGG 1 13 1 0 0 2 77961 major histocompatibility complex,class I, B GGAACTGTGA 90 84 10 18 10 2 38972 tetraspan 1 GGAAGAGCAC 2111 1 1 2 5 75268 sialyltransferase 4C (beta-galactosidasealpha-2,3-sialytransferase) GGAGGCCGAG 13 9 5 0 2 5 301342 ESTs, Weaklysimilar to GGAGGCGCTC 5 11 1 1 0 1 33455 peptidyl arginine deiminase,type II GGATGGCTTA 25 5 2 1 1 1 64179 hypothetical protein GGCACCGTGC 2244 8 10 8 4 120912 ESTs GGCCCTGCAG 14 7 1 0 5 1 105463 sir2-relatedprotein type 6 GGCTCGGGAT 15 11 2 4 3 5 2575 calpain 1, (mu/l) largesubunit GGCTGCCTGC 13 11 4 3 5 2 180958 ESTs GGCTGCCTGC 13 11 4 3 5 2197314 ESTs GGCTGGGCCT 46 25 14 5 10 8 144102 EST GGCTGGGCCT 46 25 14 510 8 14846 Homo sapiens mRNA; cDNA DKFZp564D016 (from cloneDKFZp564D016) GGCTGGGCCT 46 25 14 5 10 8 73919 clathrin, lightpolypeptide (Lcb) GGGAAGCAGA 32 17 18 4 8 9 GGGACGAGTG 20 6 1 3 6 1 3337transmembrane 4 superfamily member 1 GGGCGCTGTG 11 27 3 6 4 5 8372ubiquinol-cytochrome c reductase (6.4 kD) subunit GGGGCAGGGC 48 64 27 1015 31 119140 eukaryotic translation initiation factor 5A GGTGAAGAGG 1632 5 3 10 9 233950 serine protease inhibitor, Kunitz type 1 GTAGCAGGTG24 27 11 7 7 7 140452 cargo selection protein (mannose 6 phosphatereceptor binding protein) GTATTGGGGC 5 7 0 0 0 0 GTCATCACCA 35 22 0 0 00 107382 KIAA1517 protein GTCATCACCA 35 22 0 0 0 0 257045 Homo sapienscDNA: FLJ23415 fis, clone HEP20738 GTCATCACCA 35 22 0 0 0 0 32966guanylate cyclase activator 2B (uroguanylin) GTCATCACCA 35 22 0 0 0 068877 cytochrome b-245, alpha polypeptide GTCCGAGTGC 17 3 0 0 0 0 3337transmembrane 4 superfamily member 1 GTCCTGAACA 7 3 0 0 0 0 78546ATPase, Ca++ transporting, plasma membrane 1 GTCCTGAACA 7 3 0 0 0 0 8258DKFZP434D1335 protein GTGCACTGAG 118 48 14 7 12 13 181244 majorhistocompatibility complex, class I, A GTGCACTGAG 118 45 14 7 12 13277477 major histocompatibility complex, class I, C GTGCCTGAGA 18 15 2 67 3 77886 lamin A/C GTGGCGGGAA 3 15 1 0 4 0 GTGGGGGCGC 5 22 2 0 1 0254105 enolase 1, (alpha) GTGGTGGCAG 29 11 1 1 10 3 194691 rotinoic acidinduced 3 GTGGTTCACG 4 5 0 0 0 0 272088 ESTs, Moderately similar toGTGGTTCACG 4 5 0 0 0 0 62192 coagulation factor III (thromboplastin,tissue factor) GTGTTGGGGG 21 19 8 6 1 3 55016 hypothetical proteinFLJ21935 GTTTAGAGGG 5 16 0 0 2 1 181874 interferon-induced protein withtetratricopeptide repeats 4 TAAATTGCAA 103 59 8 17 5 3 56205 Insulininduced gene 1 TAAGGCCTTT 6 9 1 0 0 1 20149 deleted in lymphocyticleukemia, 1 TAAGGCCTTT 6 9 1 0 0 1 42945 acid sphingomyelinase-likephosphodiesterase TAATCCCAGC 37 33 8 7 16 13 TAATTTGCAT 25 2 1 1 2 079368 epithelial membrane protein 1 TACGGTGTGG 7 13 2 2 1 0 105460DKFZP56400823 protein TACTCGGCCA 10 5 0 0 1 0 79474 tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilonpolypeptide TACTGTGGAT 4 11 0 2 2 1 21537 protein phosphatase 1,catalytic subunit, beta isoform TAGACTAGCA 31 27 6 6 22 4 100090tetraspan 3 TAGGATGGGG 24 30 4 7 6 1 76941 ATPase, Na+/K+ transporting,beta 3 polypeptide TATGATGAGC 13 21 2 2 1 5 205126 Homo sapiens cDNA:FLJ22667 fis, clone HSI08385 TCACAGTGCC 26 7 3 1 3 4 81008 filamin B,beta (actin-binding protein-278) TCACCGGTCA 118 75 10 10 5 6 290070gelsolin (amyloidosis, Finnish type) TCAGAGCGCT 5 21 0 7 0 1 92323 FXYDdomain-containing ion transport regulator 3 TCAGCTGCAA 56 16 0 0 9 3284199 mucin 12 TCAGCTGCAA 56 16 0 0 9 3 301888 Homo sapiens cDNAFLJ11205 fis, clone PLACE1007843 TCGGAGCTGT 21 20 9 6 3 0 4055 Gprotein-coupled receptor kinase-interactor 1 TCTGAATTAT 24 16 0 0 1 050964 carcinoembryonic antigen-related cell adhesion molecule 1 (biliaryglycoprotein) TGACTAATTG 7 9 2 0 0 3 293380 ESTs TGAGTGACAG 9 68 0 6 3 7205126 Homo sapiens cDNA: FLJ22667 fis, clone HSI08385 TGAGTGACAG 9 68 06 3 7 271888 ESTs TGATCTCTGT 6 7 1 0 1 1 30738 hypothetical proteinFLJ10407 TGCAGCACGA 6 185 5 30 24 16 110309 major histocompatibilitycomplex, class I, F TGCAGCGCCT 16 9 1 1 1 1 77573 uridine phosphorylaseTGCCGCCCGC 14 5 2 2 1 0 202097 procollagen C-endopeptidase enhancerTGCTCCTACC 140 113 70 22 17 22 111732 Fc fragment of IgG binding proteinTGCTCCTACC 140 113 70 22 17 22 301256 Homo sapiens chromosome 19, cosmidR30669 TGGCCATCTG 30 24 8 7 3 4 184052 PP1201 protein TGGCGCGTGT 25 8 00 9 5 25640 claudin 3 TGGCTACTTA 6 9 1 0 1 2 117950 multifunctionalpolypeptide similar to SAICAR synthetase and AIR carboxylase TGGGGAGAGG43 18 20 7 3 7 288998 S100-type calcium binding protein A14 TTAACCCCTC34 14 5 9 1 5 78224 ribonuclease, RNase A family, 1 (pancreatic)TTATGGTGTG 11 17 0 0 0 0 271499 ESTs TTCCACTAAC 29 9 7 4 5 5 79706plectin 1, intermediate filament binding protein, 500 kD TTCCGCGTTC 5 160 0 2 2 137274 ESTs, Weakly similar to TTCTGGTGCG 8 2 0 0 1 0 119251ubiquinol-cytochrome c reductase core protein 1 TTCTGTAGCC 13 23 4 2 4 25541 ATPase, Ca++ transporting, ubiquitous TTGGACCTGG 33 31 7 18 12 1689761 ATP synthase, H+ transporting, mitochondrial F1 complex, deltasubunit TTGGGGTTTC 111 184 50 81 67 50 62954 ferritin, heavy polypeptide1 TTTAACGGCC 93 67 36 35 11 30 mito Tag matches mitochondrial sequenceTTTCCTCTCA 21 8 6 2 4 3 184510 stratifin TTTCCTCTCA 21 8 6 2 4 3 303400ESTs TTTCTCGTCG 10 16 2 3 0 2 1686 guanine nucleotide binding protein (Gprotein), alpha 11 (Gq class) TTTGGTTTCA 2 13 0 0 0 0 carcinoembryonicantigen-related cell adhesion molecule 1 (biliary TTTTCTGCAT 8 7 1 0 1 250964 glycoprotein) TTTTCTGCAT 8 7 1 0 1 2 77318 platelet-activatingfactor acetylhydrolase, isoform lb, alpha subunit (45 kD) TTTTTACTGA 3219 10 10 8 1 111577 integral membrane protein 2C

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1 334 1 10 DNA Homo sapiens 1 atgtaaaaaa 10 2 10 DNA Homo sapiens 2taatttttgc 10 3 10 DNA Homo sapiens 3 gtgtgtttgt 10 4 10 DNA Homosapiens 4 gtgctcattc 10 5 10 DNA Homo sapiens 5 ttccagctgc 10 6 10 DNAHomo sapiens 6 accattggat 10 7 10 DNA Homo sapiens 7 tttccactaa 10 8 10DNA Homo sapiens 8 caaggaccag 10 9 10 DNA Homo sapiens 9 aggaccatcg 1010 10 DNA Homo sapiens 10 gtcatcacca 10 11 10 DNA Homo sapiens 11ccttcaaatc 10 12 10 DNA Homo sapiens 12 tctgaattat 10 13 10 DNA Homosapiens 13 ttatggtgtg 10 14 10 DNA Homo sapiens 14 ctggcaaagg 10 15 10DNA Homo sapiens 15 aggtgactgg 10 16 10 DNA Homo sapiens 16 cttatggtcc10 17 10 DNA Homo sapiens 17 atgatggcac 10 18 10 DNA Homo sapiens 18gtccgagtgc 10 19 10 DNA Homo sapiens 19 atttcaagat 10 20 10 DNA Homosapiens 20 caagagtttc 10 21 10 DNA Homo sapiens 21 gccatcctcc 10 22 10DNA Homo sapiens 22 acccaactgc 10 23 10 DNA Homo sapiens 23 gcccacgtca10 24 10 DNA Homo sapiens 24 tttggtttca 10 25 10 DNA Homo sapiens 25ctcagaactt 10 26 10 DNA Homo sapiens 26 ccaacaccag 10 27 10 DNA Homosapiens 27 gccacatact 10 28 10 DNA Homo sapiens 28 gtattggggc 10 29 10DNA Homo sapiens 29 ccggcttgag 10 30 10 DNA Homo sapiens 30 gatatgtaaa10 31 10 DNA Homo sapiens 31 cataggttta 10 32 10 DNA Homo sapiens 32gtcctgaaca 10 33 10 DNA Homo sapiens 33 aaaagaaact 10 34 10 DNA Homosapiens 34 aacgaggaat 10 35 10 DNA Homo sapiens 35 aagaagatag 10 36 10DNA Homo sapiens 36 aataggtcca 10 37 10 DNA Homo sapiens 37 acaactcaat10 38 10 DNA Homo sapiens 38 acaactcaat 10 39 10 DNA Homo sapiens 39acatcatcga 10 40 10 DNA Homo sapiens 40 accattggat 10 41 10 DNA Homosapiens 41 acctgtatcc 10 42 10 DNA Homo sapiens 42 actccaaaaa 10 43 10DNA Homo sapiens 43 agcacctcca 10 44 10 DNA Homo sapiens 44 aggaccatcg10 45 10 DNA Homo sapiens 45 agggcttcca 10 46 10 DNA Homo sapiens 46atggctggta 10 47 10 DNA Homo sapiens 47 atgtaaaaaa 10 48 10 DNA Homosapiens 48 atgtaaaaaa 10 49 10 DNA Homo sapiens 49 atgtaaaaaa 10 50 10DNA Homo sapiens 50 attctccagt 10 51 10 DNA Homo sapiens 51 caaggaccag10 52 11 DNA Homo sapiens 52 caatattttg t 11 53 10 DNA Homo sapiens 53cagctcactg 10 54 10 DNA Homo sapiens 54 catttgtaat 10 55 10 DNA Homosapiens 55 cctagctgga 10 56 10 DNA Homo sapiens 56 ccttcgagat 10 57 10DNA Homo sapiens 57 ctcctcacct 10 58 10 DNA Homo sapiens 58 ctgacttgtg10 59 10 DNA Homo sapiens 59 ctgggttaat 10 60 11 DNA Homo sapiens 60ctgttgtatt g 11 61 10 DNA Homo sapiens 61 ctgttggtga 10 62 10 DNA Homosapiens 62 gaaaaatggt 10 63 10 DNA Homo sapiens 63 gagtcaggag 10 64 10DNA Homo sapiens 64 gcataatagg 10 65 9 DNA Homo sapiens 65 gcatttaat 966 9 DNA Homo sapiens 66 gcatttaat 9 67 10 DNA Homo sapiens 67gcatttgaca 10 68 10 DNA Homo sapiens 68 gcttttaagg 10 69 10 DNA Homosapiens 69 ggaccactga 10 70 10 DNA Homo sapiens 70 gggggtaact 10 71 10DNA Homo sapiens 71 gtgcgctgag 10 72 10 DNA Homo sapiens 72 gtgctcattc10 73 10 DNA Homo sapiens 73 gtgctcattc 10 74 10 DNA Homo sapiens 74gtgtgtttgt 10 75 10 DNA Homo sapiens 75 gttcgtgcca 10 76 10 DNA Homosapiens 76 gttcgtgcca 10 77 10 DNA Homo sapiens 77 taataaaggt 10 78 10DNA Homo sapiens 78 taatttttgc 10 79 10 DNA Homo sapiens 79 tcacaagcaa10 80 10 DNA Homo sapiens 80 tcagatcttt 10 81 10 DNA Homo sapiens 81tcctgcccca 10 82 10 DNA Homo sapiens 82 tgaaataaaa 10 83 10 DNA Homosapiens 83 tgaaataaaa 10 84 10 DNA Homo sapiens 84 tgatgtctgg 10 85 10DNA Homo sapiens 85 tgtaatcaat 10 86 10 DNA Homo sapiens 86 ttaccatatc10 87 10 DNA Homo sapiens 87 ttatgggatc 10 88 10 DNA Homo sapiens 88ttcaattaaa 10 89 9 DNA Homo sapiens 89 ttccagctc 9 90 9 DNA Homo sapiens90 ttccagctc 9 91 9 DNA Homo sapiens 91 ttccagctc 9 92 10 DNA Homosapiens 92 tttccactaa 10 93 10 DNA Homo sapiens 93 tttccactaa 10 94 10DNA Homo sapiens 94 tttttaatgt 10 95 10 DNA Homo sapiens 95 aaatctggca10 96 10 DNA Homo sapiens 96 aacgtgcagg 10 97 10 DNA Homo sapiens 97aagaaagctc 10 98 10 DNA Homo sapiens 98 aagaaagctc 10 99 10 DNA Homosapiens 99 aagaagcagg 10 100 10 DNA Homo sapiens 100 aaggtagcag 10 10110 DNA Homo sapiens 101 aataaaggct 10 102 10 DNA Homo sapiens 102aatagtttcc 10 103 10 DNA Homo sapiens 103 aatcacaaat 10 104 10 DNA Homosapiens 104 aatgagaagg 10 105 10 DNA Homo sapiens 105 acaattggtc 10 10610 DNA Homo sapiens 106 acacccatca 10 107 10 DNA Homo sapiens 107acagggtgac 10 108 10 DNA Homo sapiens 108 acattgggtg 10 109 10 DNA Homosapiens 109 acattgggtg 10 110 10 DNA Homo sapiens 110 acccaactgc 10 11110 DNA Homo sapiens 111 acccacgtca 10 112 10 DNA Homo sapiens 112acccccccgc 10 113 10 DNA Homo sapiens 113 acccccccgc 10 114 10 DNA Homosapiens 114 acctgcatcc 10 115 10 DNA Homo sapiens 115 acctggggag 10 11610 DNA Homo sapiens 116 acctggggag 10 117 10 DNA Homo sapiens 117acggtccagg 10 118 10 DNA Homo sapiens 118 actcttgttg 10 119 10 DNA Homosapiens 119 actgtggcgg 10 120 10 DNA Homo sapiens 120 agaatagctt 10 12110 DNA Homo sapiens 121 agcaggagca 10 122 10 DNA Homo sapiens 122agcaggagca 10 123 10 DNA Homo sapiens 123 agcccgacca 10 124 10 DNA Homosapiens 124 aggatggtcc 10 125 10 DNA Homo sapiens 125 aggccaaggg 10 12610 DNA Homo sapiens 126 aggtgactgg 10 127 10 DNA Homo sapiens 127agtgggctca 10 128 10 DNA Homo sapiens 128 atactccact 10 129 10 DNA Homosapiens 129 atataatctg 10 130 10 DNA Homo sapiens 130 atcgtggcgg 10 13110 DNA Homo sapiens 131 atgacgctca 10 132 10 DNA Homo sapiens 132atgatggcac 10 133 10 DNA Homo sapiens 133 atgcggagtc 10 134 10 DNA Homosapiens 134 atgcgggaga 10 135 10 DNA Homo sapiens 135 atggcacgga 10 13610 DNA Homo sapiens 136 atggtctacg 10 137 10 DNA Homo sapiens 137atggtggggg 10 138 10 DNA Homo sapiens 138 atgtgcgtgg 10 139 10 DNA Homosapiens 139 atgtgggctc 10 140 10 DNA Homo sapiens 140 atgtgggctc 10 14110 DNA Homo sapiens 141 attggagtgc 10 142 10 DNA Homo sapiens 142atttcaagat 10 143 10 DNA Homo sapiens 143 atttcaagat 10 144 10 DNA Homosapiens 144 caaataaaag 10 145 10 DNA Homo sapiens 145 caagagtttc 10 14610 DNA Homo sapiens 146 cacccctgat 10 147 10 DNA Homo sapiens 147cagtgcgttc 10 148 10 DNA Homo sapiens 148 cataggttta 10 149 10 DNA Homosapiens 149 ccaaagctat 10 150 10 DNA Homo sapiens 150 ccaacaccag 10 15110 DNA Homo sapiens 151 ccactgcacc 10 152 10 DNA Homo sapiens 152ccaggggaga 10 153 10 DNA Homo sapiens 153 ccattccact 10 154 10 DNA Homosapiens 154 cccaacgcgc 10 155 10 DNA Homo sapiens 155 cccccgaagc 10 15610 DNA Homo sapiens 156 cccccgcgga 10 157 10 DNA Homo sapiens 157ccccctgcat 10 158 10 DNA Homo sapiens 158 cccgcctctt 10 159 10 DNA Homosapiens 159 ccctcccgaa 10 160 10 DNA Homo sapiens 160 ccgctgcact 10 16110 DNA Homo sapiens 161 ccggcttgag 10 162 10 DNA Homo sapiens 162cctccagcta 10 163 10 DNA Homo sapiens 163 cctccagtac 10 164 10 DNA Homosapiens 164 cctgcccccc 10 165 10 DNA Homo sapiens 165 cctgctgcag 10 16610 DNA Homo sapiens 166 cctgcttgtc 10 167 10 DNA Homo sapiens 167cctgcttgtc 10 168 10 DNA Homo sapiens 168 cctggaagag 10 169 10 DNA Homosapiens 169 cctgtctgcc 10 170 10 DNA Homo sapiens 170 cctgtgacag 10 17110 DNA Homo sapiens 171 ccttcaaatc 10 172 10 DNA Homo sapiens 172cgaggggcca 10 173 10 DNA Homo sapiens 173 cgctgtgggg 10 174 10 DNA Homosapiens 174 cggactcact 10 175 10 DNA Homo sapiens 175 cggactcact 10 17610 DNA Homo sapiens 176 cgggagtcgg 10 177 10 DNA Homo sapiens 177cggtgggacc 10 178 10 DNA Homo sapiens 178 cgtgggtggg 10 179 10 DNA Homosapiens 179 ctaccctcac 10 180 10 DNA Homo sapiens 180 ctcagaactt 10 18110 DNA Homo sapiens 181 ctgaacctcc 10 182 10 DNA Homo sapiens 182ctgacctgtg 10 183 10 DNA Homo sapiens 183 ctggatctgg 10 184 10 DNA Homosapiens 184 ctggcaaagg 10 185 10 DNA Homo sapiens 185 ctggccctcg 10 18610 DNA Homo sapiens 186 ctggccctcg 10 187 10 DNA Homo sapiens 187ctgcccctcg 10 188 10 DNA Homo sapiens 188 ctggctatcc 10 189 10 DNA Homosapiens 189 ctgggcctct 10 190 10 DNA Homo sapiens 190 ctgtacttgt 10 19110 DNA Homo sapiens 191 ctgtgtggct 10 192 10 DNA Homo sapiens 192ctgtgtggct 10 193 10 DNA Homo sapiens 193 cttacaagca 10 194 10 DNA Homosapiens 194 cttagagggg 10 195 10 DNA Homo sapiens 195 cttatggtcc 10 19610 DNA Homo sapiens 196 cttccagcta 10 197 10 DNA Homo sapiens 197cttcttgccc 10 198 10 DNA Homo sapiens 198 cttgacatac 10 199 10 DNA Homosapiens 199 cttgattccc 10 200 10 DNA Homo sapiens 200 gacatcaagt 10 20110 DNA Homo sapiens 201 gaccagccca 10 202 10 DNA Homo sapiens 202gaccagtggc 10 203 10 DNA Homo sapiens 203 gacgcggcgc 10 204 10 DNA Homosapiens 204 gagagctccc 10 205 10 DNA Homo sapiens 205 gagcaccgtg 10 20610 DNA Homo sapiens 206 gatatgtaaa 10 207 10 DNA Homo sapiens 207gatcccaact 10 208 10 DNA Homo sapiens 208 gatgaatccg 10 209 10 DNA Homosapiens 209 gatgaccccc 10 210 10 DNA Homo sapiens 210 gcaagaaagt 10 21110 DNA Homo sapiens 211 gcacaggtca 10 212 10 DNA Homo sapiens 212gcaccctttc 10 213 10 DNA Homo sapiens 213 gcacctgtcg 10 214 10 DNA Homosapiens 214 gcacctgtcg 10 215 10 DNA Homo sapiens 215 gcagctcctg 10 21610 DNA Homo sapiens 216 gcaggaggtg 10 217 10 DNA Homo sapiens 217gcaggaggtg 10 218 10 DNA Homo sapiens 218 gcagggcctc 10 219 10 DNA Homosapiens 219 gccacatact 10 220 10 DNA Homo sapiens 220 gccacgtgga 10 22110 DNA Homo sapiens 221 gccagacacc 10 222 10 DNA Homo sapiens 222gccaggttgc 10 223 10 DNA Homo sapiens 223 gccaggttgc 10 224 10 DNA Homosapiens 224 gccaggttgc 10 225 10 DNA Homo sapiens 225 gccatcctcc 10 22610 DNA Homo sapiens 226 gcccacacag 10 227 10 DNA Homo sapiens 227gcccacgtca 10 228 10 DNA Homo sapiens 228 gcccagggcc 10 229 10 DNA Homosapiens 229 gcccagggcc 10 230 10 DNA Homo sapiens 230 gcccagggcc 10 23110 DNA Homo sapiens 231 gcccaggtca 10 232 10 DNA Homo sapiens 232gcccagtggc 10 233 10 DNA Homo sapiens 233 gccgaccagg 10 234 10 DNA Homosapiens 234 gccgggtggg 10 235 10 DNA Homo sapiens 235 gccgtggaga 10 23610 DNA Homo sapiens 236 gcctggccat 10 237 10 DNA Homo sapiens 237gcctggccat 10 238 10 DNA Homo sapiens 238 gcgaaaccct 10 239 10 DNA Homosapiens 239 gcgaaactcg 10 240 10 DNA Homo sapiens 240 gcgcagaggt 10 24110 DNA Homo sapiens 241 gctcttcccc 10 242 10 DNA Homo sapiens 242gctgcctttg 10 243 10 DNA Homo sapiens 243 gctgcccttg 10 244 10 DNA Homosapiens 244 gctggcacat 10 245 10 DNA Homo sapiens 245 gctggccccg 10 24610 DNA Homo sapiens 246 gctgtgcctg 10 247 10 DNA Homo sapiens 247gcttggggat 10 248 10 DNA Homo sapiens 248 ggaacagggg 10 249 10 DNA Homosapiens 249 ggaacagggg 10 250 10 DNA Homo sapiens 250 ggaacagggg 10 25110 DNA Homo sapiens 251 ggaactgtga 10 252 10 DNA Homo sapiens 252ggaagagcac 10 253 10 DNA Homo sapiens 253 ggaggccgag 10 254 10 DNA Homosapiens 254 ggaggcgctc 10 255 10 DNA Homo sapiens 255 ggatggctta 10 25610 DNA Homo sapiens 256 ggcaccgtgc 10 257 10 DNA Homo sapiens 257ggccctgcag 10 258 10 DNA Homo sapiens 258 ggctcgggat 10 259 10 DNA Homosapiens 259 ggctgcctgc 10 260 10 DNA Homo sapiens 260 ggctgcctgc 10 26110 DNA Homo sapiens 261 ggctgggcct 10 262 10 DNA Homo sapiens 262ggctgggcct 10 263 10 DNA Homo sapiens 263 ggctgggcct 10 264 10 DNA Homosapiens 264 gggaagcaga 10 265 10 DNA Homo sapiens 265 gggacgagtg 10 26610 DNA Homo sapiens 266 gggcgctgtg 10 267 10 DNA Homo sapiens 267ggggcagggc 10 268 10 DNA Homo sapiens 268 ggtgaagagg 10 269 10 DNA Homosapiens 269 gtagcaggtg 10 270 10 DNA Homo sapiens 270 gtattggggc 10 27110 DNA Homo sapiens 271 gtcatcacca 10 272 10 DNA Homo sapiens 272gtcatcacca 10 273 10 DNA Homo sapiens 273 gtcatcacca 10 274 10 DNA Homosapiens 274 gtcatcacca 10 275 10 DNA Homo sapiens 275 gtccgagtgc 10 27610 DNA Homo sapiens 276 gtcctgaaca 10 277 10 DNA Homo sapiens 277gtcctgaaca 10 278 10 DNA Homo sapiens 278 gtgcactgag 10 279 10 DNA Homosapiens 279 gtgcactgag 10 280 10 DNA Homo sapiens 280 gtgcctgaga 10 28110 DNA Homo sapiens 281 gtggcgggaa 10 282 10 DNA Homo sapiens 282gtgggggcgc 10 283 10 DNA Homo sapiens 283 gtggtggcag 10 284 10 DNA Homosapiens 284 gtggttcacg 10 285 10 DNA Homo sapiens 285 gtggttcacg 10 28610 DNA Homo sapiens 286 gtgttggggg 10 287 10 DNA Homo sapiens 287gtttagaggg 10 288 10 DNA Homo sapiens 288 taaattgcaa 10 289 10 DNA Homosapiens 289 taaggccttt 10 290 10 DNA Homo sapiens 290 taaggccttt 10 29110 DNA Homo sapiens 291 taatcccagc 10 292 10 DNA Homo sapiens 292taatttgcat 10 293 10 DNA Homo sapiens 293 tacggtgtgg 10 294 10 DNA Homosapiens 294 tactcggcca 10 295 10 DNA Homo sapiens 295 tactgtggat 10 29610 DNA Homo sapiens 296 tagactagca 10 297 10 DNA Homo sapiens 297taggatgggg 10 298 10 DNA Homo sapiens 298 tatgatgagc 10 299 10 DNA Homosapiens 299 tcacagtgcc 10 300 10 DNA Homo sapiens 300 tcaccggtca 10 30110 DNA Homo sapiens 301 tcagagcgct 10 302 10 DNA Homo sapiens 302tcagctgcaa 10 303 10 DNA Homo sapiens 303 tcagctgcaa 10 304 10 DNA Homosapiens 304 tcggagctgt 10 305 10 DNA Homo sapiens 305 tctgaattat 10 30610 DNA Homo sapiens 306 tgactaattg 10 307 10 DNA Homo sapiens 307tgagtgacag 10 308 10 DNA Homo sapiens 308 tgagtgacag 10 309 10 DNA Homosapiens 309 tgatctctgt 10 310 10 DNA Homo sapiens 310 tgcagcacga 10 31110 DNA Homo sapiens 311 tgcagcgcct 10 312 10 DNA Homo sapiens 312tgccgcccgc 10 313 10 DNA Homo sapiens 313 tgctcctacc 10 314 10 DNA Homosapiens 314 tgctcctacc 10 315 10 DNA Homo sapiens 315 tggccatctg 10 31610 DNA Homo sapiens 316 tggcgcgtgt 10 317 10 DNA Homo sapiens 317tggctactta 10 318 10 DNA Homo sapiens 318 tggggagagg 10 319 10 DNA Homosapiens 319 ttaacccctc 10 320 10 DNA Homo sapiens 320 ttatggtgtg 10 32110 DNA Homo sapiens 321 ttccactaac 10 322 10 DNA Homo sapiens 322ttccgcgttc 10 323 10 DNA Homo sapiens 323 ttctggtgcg 10 324 10 DNA Homosapiens 324 ttctgtagcc 10 325 10 DNA Homo sapiens 325 ttggacctgg 10 32610 DNA Homo sapiens 326 ttggggtttc 10 327 10 DNA Homo sapiens 327tttaacggcc 10 328 10 DNA Homo sapiens 328 tttcctctca 10 329 10 DNA Homosapiens 329 tttcctctca 10 330 10 DNA Homo sapiens 330 tttctcgtcg 10 33110 DNA Homo sapiens 331 tttggtttca 10 332 10 DNA Homo sapiens 332ttttctgcat 10 333 10 DNA Homo sapiens 333 ttttctgcat 10 334 10 DNA Homosapiens 334 tttttactga 10

1. A method for detection of colorectal cancer, comprising the steps of:isolating an mRNA sample from feces of a subject; detecting renaldipeptidase mRNA in said mRNA sample; comparing the amount of renaldipeptidase mRNA in said mRNA sample to amounts of renal dipeptidasemRNA in normal subjects, wherein an elevated amount of renal dipeptidasemRNA in said mRNA sample is an indicator of colorectal cancer in thesubject.
 2. The method of claim 1 further comprising the step of:identifying the patient as having colorectal cancer if an elevatedamount of renal dipeptidase mRNA in said mRNA sample is observed.
 3. Themethod of claim 1 wherein renal dipeptidase mRNA is detected by RT-PCR.4. The method of claim 1 wherein renal dipeptidase mRNA is detected byhybridization of copy mRNA to a nucleic acid array.
 5. The method ofclaim 1 wherein mRNA from one or more genes selected from the groupconsisting of Tables 3 or 5 is also detected in said mRNA sample andamount of mRNA from said one or more genes is compared to amounts innormal subjects, wherein an elevated amount of at least one of saidmRNAs selected from said group and from renal dipeptidase is anindicator of colorectal cancer in the subject.
 6. A method for detectionof colorectal cancer, comprising the steps of: isolating epithelialcells from blood of a subject; isolating an mRNA sample from theepithelial cells; detecting renal dipeptidase mRNA in said mRNA sample;comparing the amount of renal dipeptidase mRNA in said mRNA sample toamounts of renal dipeptidase mRNA in normal subjects, wherein anelevated amount of renal dipeptidase mRNA in said mRNA sample is anindicator of colorectal cancer in the subject.
 7. The method of claim 6further comprising the step of: identifying the patient as havingcolorectal cancer if an elevated amount of renal dipeptidase mRNA insaid mRNA sample is observed.
 8. The method of claim 6 wherein renaldipeptidase mRNA is detected by RT-PCR.
 9. The method of claim 6 whereinrenal dipeptidase mRNA is detected by hybridization of copy mRNA to anucleic acid array.
 10. The method of claim 6 wherein mRNA from one ormore genes selected from the group consisting of Tables 3 and 5 is alsodetected in said mRNA sample and amount of mRNA from said one or moregenes is compared to amounts in normal subjects, wherein an elevatedamount of at least one of said mRNAs selected from said group and fromrenal dipeptidase is an indicator of colorectal cancer in the subject.11. A method for detection of colorectal cancer, comprising the stepsof: contacting blood of a subject with an renal dipeptidase substrate;detecting activity of renal dipeptidase in said blood by detection ofincreased reaction product or decreased renal dipeptidase substrate;comparing the amount of activity of renal dipeptidase in blood of thesubject to that in normal subjects, wherein an elevated amount ofactivity of renal dipeptidase in the blood of the subject is anindicator of colorectal cancer in the subject.
 12. The method of claim11 wherein the renal dipeptidase substrate is labeled with a fluor. 13.The method of claim 11 wherein the renal dipeptidase substrate islabeled with a radioactive atom.
 14. The method of claim 11 wherein therenal dipeptidase substrate comprises a C-terminal D-amino acid.
 15. Amethod for detection of colorectal cancer, comprising the steps of:contacting feces of a subject with a renal dipeptidase substrate;detecting activity of renal dipeptidase in said feces by detection ofincreased reaction product or decreased renal dipeptidase substrate;comparing the amount of activity of renal dipeptidase in feces of thesubject to that in normal subjects, wherein an elevated amount ofactivity of renal dipeptidase in the feces of the subject is anindicator of colorectal cancer in the subject.
 16. The method of claim15 wherein the renal dipeptidase substrate is labeled with a fluor. 17.The method of claim 15 wherein the renal dipeptidase substrate islabeled with a radioactive atom.
 18. The method of claim 15 wherein therenal dipeptidase substrate comprises a C-terminal D -amino acid.
 19. Amethod for detection of colorectal cancer, comprising the steps of:administering to a subject an antibody which specifically binds to renaldipeptidase wherein the antibody is labeled with a moiety which isdetectable from outside of the subject; detecting the moiety in thesubject from outside of the subject, wherein an area of localization ofthe moiety within the subject but outside the proximal tubules of thekidney identifies colorectal cancer.
 20. The method of claim 19 whereinthe moiety is a fluor.
 21. The method of claim 19 wherein the moiety isa radioactive atom.
 22. The method of claim 19 wherein the moiety is acontrast agent for spiral computer tomography.
 23. A method fordetection of colorectal cancer, comprising the steps of: administeringto a subject an inhibitor of renal dipeptidase wherein the inhibitor islabeled with a moiety which is detectable from outside of the subject;detecting the moiety in the subject from outside of the subject, whereinan area of localization of the moiety within the subject but outside theproximal tubules of the kidney identifies colorectal cancer.
 24. Themethod of claim 23 wherein the moiety is a fluor.
 25. The method ofclaim 23 wherein the moiety is a radioactive atom.
 26. The method ofclaim 23 wherein the moiety is a contrast agent for spiral computertomography
 27. The method of claim 23 wherein the inhibitor bindsirreversibly to renal dipeptidase.
 28. The method of claim 23 whereinthe inhibitor binds and slowly releases from renal dipeptidase with ahalf-life of greater than 4 hours.
 29. A method for detection ofcolorectal cancer, comprising the steps of: administering to a subject asubstrate for renal dipeptidase, said substrate being labeled with adetectable moiety; isolating feces from the subject; detecting in thefeces renal dipeptidase reaction product or renal dipeptidase substratewith the detectable moiety, wherein increased product or decreasedsubstrate in the feces indicates colorectal cancer in the subject. 30.The method of claim 29 wherein the substrate is administered orally. 31.The method of claim 29 wherein the substrate is administeredintravenously.
 32. The method of claim 29 wherein the substrate isadministered rectally.
 33. A method for detection of colorectal cancer,comprising the steps of: administering to a subject a substrate forrenal dipeptidase, said substrate being labeled with a detectablemoiety; isolating blood from the subject; detecting in the blood renaldipeptidase reaction product or renal dipeptidase substrate with thedetecable moiety, wherein increased product or decreased substrate inthe blood indicates colorectal cancer in the subject.
 34. The method ofclaim 33 wherein the substrate is administered orally.
 35. The method ofclaim 33 wherein the substrate is administered intravenously.
 36. Themethod of claim 33 wherein the substrate is administered rectally.
 37. Amethod for detection of colorectal cancer, comprising the steps of:isolating an mRNA sample from feces of a subject; detecting macrophageinhibitory cytokine mRNA in said mRNA sample; comparing the amount ofmacrophage inhibitory cytokine mRNA in said mRNA sample to amounts ofmacrophage inhibitory cytokine mRNA in normal subjects, wherein anelevated amount of macrophage inhibitory cytokine mRNA in said mRNAsample is an indicator of colorectal cancer in the subject.
 38. Themethod of claim 37 further comprising the step of: identifying thepatient as having colorectal cancer if an elevated amount of macrophageinhibitory cytokine mRNA in said mRNA sample is observed.
 39. The methodof claim 37 wherein macrophage inhibitory cytokine mRNA is detected byRT-PCR.
 40. The method of claim 37 wherein macrophage inhibitorycytokine mRNA is detected by hybridization of copy mRNA to a nucleicacid array.
 41. The method of claim 37 wherein mRNA from one or moregenes selected from the group consisting of Tables 3 and 5 is alsodetected in said mRNA sample and amount of mRNA from said one or moregenes is compared to amounts in normal subjects, wherein an elevatedamount of at least one of said mRNAs selected from said group and frommacrophage inhibitory cytokine is an indicator of colorectal cancer inthe subject.
 42. A method for detection of colorectal cancer, comprisingthe steps of: isolating epithelial cells from blood of a subject;isolating an mRNA sample from the epithelial cells; detecting macrophageinhibitory cytokine mRNA in said mRNA sample; comparing the amount ofmacrophage inhibitory cytokine mRNA in said mRNA sample to amounts ofmacrophage inhibitory cytokine mRNA in normal subjects, wherein anelevated amount of macrophage inhibitory cytokine mRNA in said mRNAsample is an indicator of colorectal cancer in the subject.
 43. Themethod of claim 42 further comprising the step of: identifying thepatient as having colorectal cancer if an elevated amount of macrophageinhibitory cytokine mRNA in said mRNA sample is observed.
 44. The methodof claim 42 wherein macrophage inhibitory cytokine mRNA is detected byRT-PCR.
 45. The method of claim 42 wherein macrophage inhibitorycytokine mRNA is detected by hybridization of copy mRNA to a nucleicacid array.
 46. The method of claim 42 wherein mRNA from one or moregenes selected from the group consisting of Tables 3 and 5 is alsodetected in said mRNA sample and amount of mRNA from said one or moregenes is compared to amounts in normal subjects, wherein an elevatedamount of at least one of said mRNAs selected from said group and frommacrophage inhibitory cytokine is an indicator of colorectal cancer inthe subject.
 47. A method for detection of colorectal cancer, comprisingthe steps of: detecting macrophage inhibitory cytokine in blood of asubject and comparing the amount of macrophage inhibitory cytokine inblood of the subject to that in normal subjects, wherein an elevatedamount of macrophage inhibitory cytokine in the blood of the subject isan indicator of colorectal cancer in the subject.
 48. A method fordetection of colorectal cancer, comprising the steps of: detectingmacrophage inhibitory cytokine in feces of a subject; and comparing theamount of macrophage inhibitory cytokine in feces of the subject to thatin normal subjects, wherein an elevated amount of macrophage inhibitorycytokine in the feces of the subject is an indicator of colorectalcancer in the subject.
 49. A method for detection of colorectal cancer,comprising the steps of: detecting renal dipeptidase in blood of asubject; and comparing amount of renal dipeptidase in blood of thesubject to the amount of renal dipeptidase in normal subjects, whereinan elevated amount of renal dipeptidase in the blood of the subject isan indicator of colorectal cancer in the subject.
 50. A method fordetection of colorectal cancer, comprising the steps of: detecting renaldipeptidase in feces of a subject; and comparing amount of renaldipeptidase in feces of the subject to the amount of renal dipeptidasein normal subjects, wherein an elevated amount of renal dipeptidase inthe feces of the subject is an indicator of colorectal cancer in thesubject.